Project 2: Nuclear export and translation Summary Transport of HIV-1 RNAs from the nucleus and their regulated expression in the cytoplasm are critical steps of the viral lifecycle. Domains present in viral RNA have been shown to regulate these events; namely, the export of unspliced transcripts requires the Rev responsive element, RRE; alternative conformers of the 5?-leader region dictate translation vs. packaging fates; and the recoding of genetic information that allows the production of fixed ratios of viral proteins relies on the frameshifting signal. While elucidation of these RNA structures, and thus mechanisms regulating these events, are closer to realization, studies carried out in CRNA 1.0 have established that both nuclear export and translational regulation are more multifaceted than previously thought. Several major discoveries were not previously predicted or envisioned: the cytoplasmic Gag:RRE interaction; the presence of transcriptional start site heterogeneity, and its consequence on maintaining separate pools of 5?-leader structures; and the presence of a structural equilibrium between an inactive stem-loop and an active, frameshift-permissive pseudoknot conformation. Based on these CRNA discoveries, this project aims to gain a complete structural and mechanistic understanding of both nuclear transport and translational regulation in HIV-1 by combining structural studies with visualization, biochemical and virologic experiments. Our aims for understanding nuclear export will be to determine the high resolution structures of the RRE nuclear export signal, the biological significance of Gag:RRE interactions, and the nuclear export properties of various HIV-1 RNAs. Our aims for understanding translational regulation will be to determine structures for the monomeric and the spliced env mRNA forms of the 5?-leader, and the structures involved in the process of programmed ribosomal frameshifting. !